The transgenic animal carries a gene introduced to its own or progenitor's germ cell line in an early stage of development (usually, single cells).
Wagner et al. (1981, ProNAS, USA, 78, 50169 and Stewart et al. (1982, Science, 217, 1046) describe transgenic mice carrying the human globin gene. Constantini et al. (1981, Nature, 294, 92) and Lacy et al. (1983, Cell, 34, 343) describe transgenic mice carrying the rabbit globin gene. McKnight et al. (1983, Cell, 34, 335) describe mice carrying a foreign transferrin gene. Brinstar et al. (1983, Nature, 306, 332) describe mice carrying a functionally introduced immunoglobulin gene.
With regard to transgenic mice carrying a foreign gene related to diseases of bone, there is the following report. Lewis, D. B. et al. (1993, ProNAS, USA, 90, 11618) describe mice carrying the mouse lck-IL4 gene introduced and report that the osteocalcin level in those mice was significantly low, with the animals manifesting osteoporosis-like symptoms.
Meanwhile, as mice carrying a foreign gene related to diseases of the kidney, the following reports are available. Doi, T. et al. (1988, Am. J. Pathol., 131, 398) describe mice carrying the bovine growth hormone gene introduced and report that those mice showed diffuse mesangial hyalinization at week 7 of age and advanced glomerulosclerosis at week 30 or later, and subsequently died of uremia.
Dressler, G. R. et al. (1993, Nature, 362, 65) describe mice carrying the Pax-2 gene introduced and report that, in their kidneys, expression of the gene could be confirmed and nephrotic symptoms such as glomerular atrophy and proteinuria were observed. Lowden, D. A. et al. (1993, J. Lab. Clin. Med., 124, 386) describe mice carrying a foreign TGF-.alpha. gene introduced and report that expression of the gene in the kidneys could be confirmed and such symptoms as cyst formation and glomerular hypertrophy were observed.
However, there is not known a rat carrying any foreign gene introduced which can serve as a model of bone disease or kidney disease.
It is known that vitamin D exists in the natural kingdom as two native forms, D.sub.2 and D.sub.3, and that whereas D.sub.2 has a double bond at the 22-position and a methyl group at the 24-position of its side chain and occurs in plants, D.sub.3 occurs in animals. As the 7-hydrocholesterol (provitamin D.sub.3) produced in the human skin is exposed to ultraviolet light from 290 to 320 nm, it undergoes photodegradation involving cleavage of the B ring at the 9, 10-position to give provitamin D.sub.3. This, in turn, is isomerized at body temperature to give vitamin D.sub.3. The structure of vitamin D.sub.3 contains 3 double bonds, resulting from cleavage of the B ring, in every other positions. Vitamin D.sub.3 is coupled to a vitamin D-binding protein and the couple is transported to the liver, where the 25-position of its side chain is hydroxylated by the enzyme 25-hydroxylase present in the liver cell mitochondria to give 25-hydroxyvitamin D.sub.3. This substance is thence transported to the kidneys and, depending on the calcium metabolism regulating hormones in the bloodstream, such as PTH, and the blood calcium concentration, it is further hydroxylated at the 1.alpha.-, 23-, 24- and 26-positions in the proximal renal tubules to give 1.alpha.,25-dihydroxyvitamin D.sub.3, 23,25-dihydroxyvitamin D.sub.3, 24,25-dihydroxyvitamin D.sub.3 and 26,25-dihydroxyvitamin D.sub.3, respectively. Among them, the metabolite having high biological activity is 1.alpha.,25-dihydroxyvitamin D.sub.3, which elevates blood calcium and phosphorus levels and discharges bone metabolizing functions; namely it assists in the expression of osteopontin and osteocalcin by osteoblasts, inhibits production of proteoglycan and, conversely, enhances production of cell membrane-derived phospholipids to thereby accelerate calcification of the osteoblasts. Regarding the function of 24,25-dihydroxyvitamin D.sub.3, this substance is known to inhibit formation of osteoclasts, promote expression of the osteocalcin gene and further reportedly accelerate calcification in vitamin D-deficient rats but this action is dependent on the 25-hydroxyvitamin D.sub.3 24-hydroxylase (also called vitamin D.sub.3 24-hydroxylase or 1.alpha.,25-dihydroxyvitamin D.sub.3 -hydroxylase) which is present in the proximal renal tubules. Furthermore, recent studies have revealed that this enzyme hydroxylates not only the 24-position but also the 26-position.
Among important known disorders of vitamin D metabolism are vitamin D-dependent disease Type II, rickets, osteomalacia, and so forth. Those diseases manifest dysosteogenesis and deformity of bone resulting from a disorder of calcification, and the occurrence of this disorder prior to closure of the epiphyses leads to rickets, while occurrence of the disorder at later times leads to osteomalacia. Aside from the above diseases, there exist risks for renal diseases inclusive of renal insufficiency, secondary hyperparathyroidism and hypercalcemia occurring either as complications of said diseases or independently.
Ohyama et al. (1989, FEBS Lett., 255, 405) succeeded in the purification of vitamin D.sub.3 24-hydroxylase from rat kidney mitochondria extracted after administration of vitamin D. Subsequently Ohyama et al. (1991, FEBES Lett., 278, 195) isolated a cDNA by the screening of clones using an anti-vitamin D.sub.3 24-hydroxylase antibody. It is known that this vitamin D.sub.3 24-hydroxylase cDNA has a full length of 3.2 K bases and contains a reading frame (generally called open reading frame) of 1542 bp for the translation of 514 amino acids and produces a protein having a molecular weight of 59,000. It has been shown that as 35 amino acids are cleaved off from the N-terminus of the above protein, a mature protein consisting of 479 amino acid residues and having a molecular weight of about 55,000 is produced. Moreover, the finding that its 462nd amino acid cysteine is bound to the 5-position of heme indicates that this protein has characteristics similar to those of mitochondrial protein P-450, and an expression experiment in COS cells gave evidence of expression as a protein. Meanwhile, human vitamin D.sub.3 24-hydroxylase cDNA has been isolated by Chen et al. (1993, ProNAS, USA, 90, 4543), the murine counterpart by Itoh et al. (1995, Biochemica et Biophysica Acta, 1264, 26), and guinea pig counterpart by Ohyama et al. (1996, Journal of Japan Society of Bone Metabolism, 14, 112). It has been shown that the amino acid sequences of the above rat, human, mouse and guinea pig versions of the enzyme have about 80 to 95% homology.
The functions of the enzyme in vivo has also been analyzed, and Shinki et al. (1992, J. Biol. Chem., 267, 13757) demonstrated by Northern analysis that vitamin D.sub.3 24-hydroxylase is induced by 1.alpha.,25-dihydroxyvitamin D.sub.3. It was also shown that the reaction of 1.alpha.,25-dihydroxyvitamin D.sub.3 is faster and higher in degree in the small intestine than in the kidneys. Furthermore, in the analysis of the relative reactivity of 25-hydroxyvitamin D.sub.3 and 1.alpha.,25-dihydroxyvitamin D.sub.3, the difference in Km value suggested that 1.alpha.,25-dihydroxyvitamin D.sub.3 is higher in the substrate specificity of vitamin D.sub.3 24-hydroxylase. Bechen, M. J. et al. (1996, Biochemistry, 35, 8465) investigated the metabolism of 25-hydroxyvitamin D.sub.3 experimentally in Spodoptera frugiperda (Sf21) cells and suggested that the enzyme has 23-/24-bicatalytic activity. Furthermore, vitamin D.sub.3 25-hydroxylase has been isolated from rat liver mitochondria by Masumoto et al. (1988, J. Biol. Chem., 263, 14256) and some light has been cast on the pertinent gene function as well. However, neither isolation and purification nor cloning of vitamin D.sub.3 1.alpha.-hydroxylase gene has been successful to this day.
Expression of vitamin D.sub.3 24-hydroxylase gene is regulated by the coupling of the heterodimer consisting of the vitamin D.sub.3 receptor (generally abbreviated as VDR)-1.alpha.,25-dihydroxyvitamin D.sub.3 complex and the retinoid X receptor (generally abbreviated as RXR) to the vitamin D.sub.3 response element (generally abbreviated as VDRE) VDRE.sub.1 located in the -150 to -136 region of the 5'-upstream. This vitamin D.sub.3 response element has a repetitive structure of a motif consisting of the 6 bases of AGGTCA with 3 base gaps (generally called tandem repeat) and its similarity to the thyroid hormone response element (generally abbreviated as TRE) and retinoic acid response element (generally abbreviated as RARE) has been pointed out. Furthermore, Kerry, D. M. et al. (1996, J. Biol. Chem., 271, 29715) suggested that among the three vitamin D.sub.3 response elements existing in the 5'-upstream of this gene, VDRE-1 which is situated in the -150 to -136 region is more sensitive to 1.alpha.,25-dihydroxyvitamin D.sub.3 than is VDRE-2 which is situated in the -249 to -232 region and that those two elements cooperates to enhance the activity and modulate the response to 1.alpha.,25-dihydroxyvitamin D.sub.3.
The important genes under up regulation of expression by 1.alpha.,25-dihydroxyvitamin D.sub.3 include alkaline phosphatase, aldolase subunit B, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein-70, calbindin-D.sub.28K and .sub.9K, osteocalcin, osteopontin, osteonectin, fibronectin, interleukin I-6, matrix-gla-protein, metallothionein, NADH-DH subunit III and IV, integrin .alpha.V.beta.3, transforming growth factor .beta., nerve growth factor, c-FMS, c-fos, c-Ki-ras, vitamin D.sub.3 receptor, 25-hydroxyvitamin D.sub.3 24-hydroxylase, protein kinase C, prolactin, plasma membrane calcium pump, EGF receptor, tumor necrosis factor .alpha., 1.alpha.,25-dihydroxyvitamin D.sub.3 receptor, etc. The important genes under down regulation of expression include NADH-DH subunit I, calcitonin, collagen type I, .gamma.-interferon, colony stimulating factor, c-myb, 25-hydroxyvitamin D.sub.3 1.alpha.-hydroxylase, fatty acid-binding proteins, interleukin II and III, CD-23, transferrin receptor, cytochrome B, ferredoxin, parathyroid hormone (generally abbreviated as PTH), prepro-PTH, PTH-related proteins, protein kinase inhibitors, etc. It is known that the vitamin D.sub.3 24-hydroxylase, osteocalcin and osteopontin regulatory regions respectively have vitamin D.sub.3 -response elements and that the expression of those proteins is regulated by 1.alpha.,25-dihydroxyvitamin D.sub.3 but it remains to be known as yet whether all of the above-enumerated genes are under the regulation through the vitamin D.sub.3 -response elements.
As 1.alpha.,25-dihydroxyvitamin D.sub.3 was administered to a rat with vitamin D.sub.3 deficiency, vitamin D.sub.3 24-hydroxylase was induced but its concentration and reaction time varied between the kidney and the small intestine, with a higher reactivity being found in the small intestine. It was shown that concurrent administration of parathyroid hormone and 1.alpha.,25-hydroxylated vitamin D.sub.3 inhibited the induction of vitamin D.sub.3 24-hydroxylase. It was also shown that, in the kidneys, this enzyme is expressed in the proximal tube. Roy et al. (1996, Endocrinology, 137, 2938) demonstrated that, in the small intestine, this 1.alpha.,25-dihydroxyvitamin D.sub.3 -induced enzyme is expressed in the saccular gland columnar epithelium and villi.
Regarding vitamin D, reactions due to gene activation (generally called the genomic action of vitamin D) and other reactions (generally called the non-genomic action of vitamin D) are known and have been suggested to be associated with different physiological activities. As to the non-genomic action of vitamin D, there are mentioned such phenomena as promotion of the intestinal uptake of calcium and induction of an increase in intracellular calcium in a matter of a few minutes.
The metabolites of vitamin D in human plasma vary with assay conditions but taking vitamin D.sub.3 as an example, reportedly its normal range in plasma is 1 to 5 ng/ml with an elimination half-life of 1 day; in the case of 25-hydroxyvitamin D.sub.3 its normal range is 10 to 40 ng/ml with an elimination half-life of 10 to 20 days; in the case of 24-hydroxyvitamin D.sub.3 its normal range is 1 to 4 ng/ml with an elimination half-life of 14 to 21 days; and in the case of 1.alpha.,25-dihydroxyvitamin D.sub.3 its normal range is 20 to 70 pg/ml with an elimination half-life of several hours. 25-Hydroxyvitamin D.sub.3 is produced in the liver, and this substance is little subject to metabolic regulation but is dependent on vitamin D intake and photobiosynthesis and can, therefore, be a nutritional marker of vitamin D deficiency. On the other hand, 1.alpha.,25-dihydroxyvitamin D.sub.3 is regulated by the blood calcium concentration and-parathyroid hormone level and metabolized in the kidneys so that it is maintained at a constant concentration. Low plasma levels of this substance are found in certain diseases such as vitamin D-dependent disease type II, rickets, osteomalacia, chronic renal insufficiency, hypoparathyroidism, hyperthyroidism and osteoporosis, while high plasma levels are found in such diseases as secondary parathyroidism and hypercalcemia and in pregnancy. Thus, it is known that this substance can be a diagnostic marker of those diseases.
Nutritionally speaking, the intake of foods or preparations containing vitamin D.sub.2 and vitamin D.sub.3 is effective in vitamin D deficiency but administration of an activated vitamin D preparation is needed in vitamin D-resistant rickets, osteomalacia, osteoporosis, renal dystrophy, psoriasis and antineoplastic drug-induced rickets.
The synthesis of activated vitamin D derivatives is going on with avidity and a large number of derivatives have been synthesized. In line with this development, advances have been made in the study of the biological activity in cells and clinical effects of those derivatives as well, with the result that various vitamin D preparations are being used as therapeutic drugs for said diseases and even a large number of candidate derivatives for such therapeutic drugs are already known. Particularly, as epitomized by the report of Bouillon et al. (1995, Endocrine Reviews, 16, 200), many structure-activity relation studies have also been undertaken. Studies on the biological actions in cells and clinical effects of those substances have also made a rapid progress and, as a result, many vitamin D preparations are being used in the therapy of the above-mentioned diseases and a large number of derivatives are now earmarked as candidate therapeutic drugs.
Among them, 24-fluorinated vitamin D.sub.3 has been studied in particular detail and as it was elucidated that the biological activity of 24,24-difluoro-25-hydroxyvitamin D.sub.3 is not different from that of 24,25-hydroxyvitamin D.sub.3, the function of 1.alpha.,25-dihydroxyvitamin D.sub.3 24-hydroxylase has come to be explored in greater depth. Beckman et al. (1996, Biochemistry, 35, 8465) demonstrated the multi-catalytic activity of this enzyme in an in vitro metabolism experiment. Thus, 25-hydroxyvitamin D.sub.3 was metabolized to 25-hydroxy-24-oxovitamin D.sub.3, to 24-oxo-23,25-hydroxyvitamin D.sub.3 and further to 23-hydroxy-24,25,26,27-tetranorvitamin D.sub.3, indicating that the activity of this enzyme is not limited to that of 24-hydroxylation but is multi-catalytic.